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Originally published In Press as doi:10.1074/jbc.M504058200 on July 25, 2005

J. Biol. Chem., Vol. 280, Issue 38, 32835-32842, September 23, 2005
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Expression of an Uncleavable N-terminal RasGAP Fragment in Insulin-secreting Cells Increases Their Resistance toward Apoptotic Stimuli without Affecting Their Glucose-induced Insulin Secretion*

Jiang-Yan Yang{ddagger}, Joël Walicki{ddagger}, Amar Abderrahmani{ddagger}, Marion Cornu§, Gérard Waeber{ddagger}, Bernard Thorens§, and Christian Widmann{ddagger}1

From the {ddagger}Department of Cellular Biology and §Department of Physiology, Faculty of Biology and Medicine, Lausanne University, Bugnon 9, Lausanne 1005, Switzerland and the Department of Internal Medicine, Lausanne University Hospital, Lausanne 1011, Switzerland

Received for publication, April 14, 2005 , and in revised form, July 5, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis of pancreatic {beta} cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of {beta} cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-{kappa}B-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-{kappa}B activity, and protects the conditionally immortalized pancreatic insulinoma {beta}TC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects {beta} cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis appears to be a critical determinant in the development of both type 1 and type 2 diabetes. Type 1 diabetes is a direct consequence of an autoimmune attack on pancreatic islet {beta}-cells leading to their death (1). Analyses of pancreatic tissues from deceased human subjects has indicated that {beta} cell mass is decreased in type 2 diabetes as well and that the mechanism underlying this is an increase in {beta} cell apoptosis (2). The higher apoptotic rate observed in {beta} cells of diabetic patients could result from deregulated levels of various circulating fuel molecules (e.g. glucose and saturated fatty acids) and chronic activation of the innate immune system (3).

Apoptosis is induced when members of the caspase family of proteases are activated. These enzymes cleave a subset of cellular proteins (4, 5), inducing the characteristic biochemical and morphological features of apoptosis. Cells can activate a series of regulatory mechanisms to maintain an adequate balance between pro- and antiapoptotic signals. For example, the ratio of proapoptotic versus anti-apoptotic Bcl-2 family members can determine whether or not a cell survives (6). Many intracellular signaling pathways also regulate cell death (7). However, the activation of a given intracellular signaling pathway does not necessarily generate the same response in different cell types. For example, activation of NF-{kappa}B is protective in fibroblasts and T cells (8, 9) but favors cells death in pancreatic {beta} cells (10, 11). The cellular context will probably modulate the way a given signaling pathway in a given cell type regulates apoptosis.

RasGAP, a regulator of Ras and Rho, is a caspase-3 substrate that functions as a sensor of caspase-3 activity in cells (12). RasGAP is cleaved in a stepwise manner as caspase activity increases. At low caspase-3 activity, RasGAP is cleaved only once at position 455, generating an N-terminal fragment, called fragment N, that induces a potent antiapoptotic Ras-phosphatidylinositol 3-kinase-Akt-dependent pathway (13, 14). This protective pathway occurs independently of NF-{kappa}B activation, since fragment N inhibits the ability of Akt to stimulate the NF-{kappa}B pathway (14). Generation of fragment N is crucial for cell survival in low stress conditions (15). At higher caspase activity, fragment N is further processed into two additional fragments, called fragments N1 and N2. Cleavage of fragment N abrogates its protective functions, and hence the second cleavage of RasGAP promotes apoptosis (24).

Pancreatic {beta} cells undergo apoptosis in response to a variety of stimuli, including nutrient deprivation and inflammatory cytokines. Counteracting the proapoptotic effects of caspases would therefore be advantageous to render {beta} cells more resistant to a series of noxious stimuli. To determine whether fragment N has a beneficial function in insulin-secreting cells, we have assessed here whether an uncleavable form of this fragment renders the conditionally immortalized pancreatic insulinoma {beta}TC-tet cell line more resistant to a series of adverse stimuli. We also compared the protection efficacy of fragment N with that of another antiapoptotic protein, Bcl-2, which has been shown to induce survival signals in {beta} cells (16) but does not activate the Ras-phosphatidylinositol 3-kinase-Akt pathway and therefore protects cells differently from fragment N (6, 17). Finally, we have determined whether the combined expression of fragment N and Bcl-2 provides additive levels of protection in {beta} cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—HA-D157A.dn3 encodes a RasGAP mutant that cannot be cleaved at position 157. Two plasmids bearing the uncleavable form of fragment N have been used for the production of fragment N-encoding lentivirus. The first one, N-D157A.lti, bears the fragment N cDNA under the control of the phosphoglycerate kinase promoter and has been described earlier (15). The second one, N-D157A.irs, has been generated by subcloning the BamHI-XhoI fragment of N-D157A.dn3 (14) in TRIP-PGK-IRESNEO-WHV opened with BamHI and SalI. This plasmid bears the neomycin resistance gene and the fragment N cDNA separated by an internal ribosomal entry site under the control of the phosphoglycerate kinase promoter. The plasmid used for the generation of Bcl-2-encoding lentivirus (SIN-PGK-hBcl-2-WHV) has been described previously (16). The plasmid encoding the dominant negative kinase-dead mutant of Akt (HA-Akt1(K179M).cmv; previously called Akt-DN.cmv) has been described earlier (14).

Lentivirus—Recombinant lentiviruses were produced as described (18). Briefly, 293T cells were co-transfected using the calcium phosphate DNA precipitation method (19) with 10 µg of the lentiviral vector containing the cDNA of interest (e.g. N-D157A.lti), 2.5 µg of the envelope protein-coding plasmid (pMD.G), and 7.5 µg of the packaging construct (pCMVDR8.91). Two days after the transfection, the virus-containing medium was harvested. To determine how much of the virus preparations was needed to infect 100% of the cells, {beta}TC-tet and {beta}TC-tet/Bcl-2 seeded at a 50% confluence on coverslips placed in 6-well plates were cultured overnight with various volumes of fragment N-encoding lentiviruses. After removal of the virus solution, the cells were maintained for 2 more days before fixation and immunocytochemical staining with antibodies directed at the HA tag. The lowest volumes of the lentiviral preparations required to infect 100% of the cells were chosen for further experiments.

Cell Lines—{beta}TC-tet and {beta}TC-tet/Bcl-2 cells (16) were maintained in Dulbecco's modified Eagle's medium (catalog number D-5671; Sigma) containing 15% decomplemented horse serum (catalog number H-1270; Sigma), 2.5% fetal calf serum (catalog number F-7524; Sigma), 10 mM HEPES (catalog number H-3537; Sigma), 2 mML-glutamine (catalog number G-7513; Sigma), 1 mM sodium pyruvate (catalog number S-8636; Sigma) at 37 °C and 5% CO2. Generation of fragment N-producing cell lines was obtained following infection with fragment N-encoding viruses in conditions leading to 100% infection efficiency (see "Lentivirus"). Generation of control cell lines was performed using empty lentiviruses using the same volume loads as when cells were infected with fragment N-encoding viruses. When viruses constructed with plasmids derived from the TRIP-PGK-IRESNEO-WHV vector were used (e.g. N-D157A.irs), a selection step with 1 mg/ml neomycin for 14 days was additionally performed. Growth arrest of the {beta}TC-tet and their derivatives was achieved by incubating the cells for 4 days with 1 µg/ml tetracycline (Fluka, Buchs, Switzerland) (16). This represses the expression of the large T antigen and prevents the cells from proliferating (20).

Chemicals and Antibodies—The anti-phosphoserine 473-Akt rabbit polyclonal IgG antibody was from Cell Signaling Technology (catalog number 9271). The rabbit polyclonal IgG antibody recognizing Akt1/2 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (catalog number SC-8312). The anti-RasGAP antibody is directed at the Src homology domains of RasGAP and has been described before (21). Anti-Bcl-2 antibody was from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (catalog number 05 341). Cisplatin was from Sigma (catalog number P4394). Cytokines (TNF{alpha},2 interleukin-1{beta}, and interferon-{gamma}) were from ALEXIS (catalog number ALX-520-002-C010, ALX-520-001-C010, and PBL-11500-2, respectively). The antibody directed at the SV40 large T antigen was from BD Biosciences (catalog number 55414).

Apoptosis Assay—The percentage of apoptosis was determined by scoring cells displaying pycnotic nuclei (visualized with Hoechst 33342) (13). Apoptosis scoring of primary islets cells was performed after dissociating the agglomerated cells in 300 µl of cell dissociation PBS-based buffer (catalog number 13151-014; Invitrogen) at 37 °C for 10–15 min. The cells were then washed once in PBS before being resuspended in 100 µl of PBS containing 10 µg/ml Hoechst 33342. Scoring of apoptosis was performed on 20 µl of the stained cells deposited on slides and covered with coverslips.

NF-{kappa}B Activity Assay—Cells were transfected with 0.5 µg of the NF-{kappa}B reporter plasmid prLUC (22) and 0.5 µg of pRL-TK encoding the Renilla luciferase (Promega) used as an internal control. One day following transfection, the cells were lysed in "passive lysis buffer" (Promega kit number E1910). Measurement of the NF-{kappa}B activation was then performed with a Promega kit (catalog number E1910) using lysates (25 µg of protein) according to the manufacturer's protocol (available on the World Wide Web at www.promega.com/tbs/tm040/tm040.pdf). The activity of the NF-{kappa}B reporter plasmid was normalized to the activity of the internal control.

Western Blot Analysis—Cells were lysed in monoQ-c buffer (13) in which 1 mM Na3VO4 was freshly added. Western blotting was performed as described previously (23, 24) using a homemade ECL reagent (13).

Immunocytochemistry—The functional infectivity of the virus preparations was determined by immunocytochemistry. Subconfluent {beta}TC-tet or CDM3D cells seeded on coverslips in 6-well plates were cultured overnight with various volumes of fragment N-encoding recombinant virus. After removal of the virus solution, the cells were maintained in culture for an additional 2 days. The next steps were performed at room temperature. The cells on coverslips were washed with 4 ml of PBS; fixed with 3 ml of PBS, 3% formaldehyde, 3% sucrose for 20 min; washed three times with PBS; incubated with 2 ml of PBS, 0.2% Triton X-100 for 10 min; washed once with PBS; and incubated 15 min at room temperature with 3 ml of filtered serum-containing culture medium. The cells were then incubated 30 min in this medium in the presence of a 1:50 dilution of an anti-HA antibody prepared as described (13). After three washes with 4 ml of PBS, the cells were incubated as above with an anti-mouse Cy3-labeled antibody (catalog number 715-165-151; Jackson ImmunoResearch) at a 1:500 dilution. After three more washes over a period of 60 min, the coverslips were mounted in Vectashield mounting medium (catalog number H-1000; Vector Laboratories, Ltd.) and visualized with a Zeiss Axioplan 2 imaging microscope equipped with a Plan-Neofluar x40/01.3 oil {infty}/0.17 lens and a Zeiss AxioCam HRC camera using the Zeiss AxioVision acquisition software.

Giemsa Staining—The cells were seeded in 6-well plates at a density of 200 cells/well. After 2 weeks, the colonies were stained as follows. The wells were washed with 5 ml of PBS and air-dried. The wells were then incubated for 15 min with ethanol and air-dried again and then incubated for 15 min with 4 ml of a Giemsa stain solution (Giemsa blood stain solution; J.T. Baker Inc.) diluted 1:5 in methanol. The stain solution was then aspirated, and the plates were placed upside down on water for at least 20 min. The plates were finally extensively washed with tap water and air-dried. Colonies were counted with a Bio-Rad Fluor-S MultiImager using the Personal Molecular Imager FX colonies counting program.

Cell Counting—Cells in 6-well plates (200 cells/well) were trypsinized for 3 min in 500 µl of 1x trypsin-EDTA solution (catalog number T3924; Sigma), followed by the addition of 500 µl of culture medium. The cells were then harvested, centrifuged at 700 x g for 5 min, and resuspended in 100 µl of culture medium in the presence of 10 µl of trypan blue solution (0.4%) (catalog number T8154; Sigma). Living cells excluding the dye were scored using a Neubauer improved counting chamber (Blue Brand, catalog number MAR-0610710).



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  		FIGURE 1.
Expression of fragment N in insulinoma {beta}TC-tet cells following lentiviral infection. A, {beta}TC-tet cells were infected with the indicated quantities of HA-tagged fragment N-encoding lentiviruses that do (lower row) or do not(upper row) co-express the neomycin resistance gene. The expression levels of fragment N were assessed by immunocytochemistry analysis 72 h after the infection using an anti-HA antibody and an anti-mouse IgG Cy3-labeled antibody. All pictures were taken with a x40 objective and with an exposure time of 4 s using a Zeiss Axiovision microscope. B, {beta}TC-tet cells and {beta}TC-tet/Bcl-2 cells were infected as described in A. The cells were then lysed, and expression of fragment N was assessed by Western blot using an anti-RasGAP antibody.

 
Insulin Quantitation—Cells were extracted with acid/ethanol (lysis in 400 µl of 75% ethanol, 1.5% concentrated hydrochloric acid for 24 h at 4 °C followed by a centrifugation at 700 x g for 3 min). Insulin content of the cell extracts was assessed using a Linco enzyme-linked immunosorbent assay kit according to the manufacturer's protocol (Linco; enzyme-linked immunosorbent assay kit, catalog numbers EZRMI-13K and E6013).

Insulin Secretion Assays—Growth-arrested cells were washed three times with a modified Krebs-Ringer/bicarbonate-HEPES buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2mM NaHCO3, 10mM HEPES, 0.1% bovine serum albumin, pH 7.4) and preincubated with Krebs-Ringer/bicarbonate-HEPES buffer containing 2 mM glucose for 1 h at 37 °C. Cells were then incubated for 35 min in Krebs-Ringer/bicarbonate-HEPES buffer, 2 mM glucose (basal response), or Krebs-Ringer/bicarbonate-HEPES buffer, 20 mM glucose, 10 µM forskolin (catalog number F-6886; Sigma), 100 µM isobutylmethylxanthine (catalog number I-7018; Sigma) (stimulated response). The supernatant was harvested for the measurement of secreted insulin, and the remaining cells were extracted with acid/ethanol for the measurement of the cellular insulin content, as described above.



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  		FIGURE 2.
Fragment N induces Akt activation and NF-{kappa}B inhibition in insulin-secreting cells. A, half a million {beta}TC-tet (–) or {beta}TC-tet/Bcl-2 cells (+) stably infected with the indicated viruses were cultured in 6-well plates for 24 h and starved in Dulbecco's modified Eagle's medium for an additional 48-h period to reduce basal Akt levels. The cells were then lysed in 150 µl of monoQ-c lysis buffer. The levels of active Akt were then assessed by Western blot analysis using an antibody specific for the phosphorylated active form of Akt. The expression levels of Akt were also assessed by Western blot using an antibody recognizing all forms of Akt (total Akt). B, {beta}TC-tet and {beta}TC-tet/Bcl-2 cells (0.5 x 106) stably infected with the indicated viruses were placed in 6-well plates. The following day, the cells were transfected with prLUC and pRL-TK plasmids, and 24 h later the cells were lysed and the NF-{kappa}B activity was measured as described under "Materials and Methods."

 
Preparation of Rat and Mouse Islets—Wistar rats (200–350 g) were killed in a CO2 atmosphere for 4 min. The abdominal cavity was then exposed, and the proximal common bile duct was occluded with a Silkan thread (BBraun, Sempach, Switzerland, catalog number 01134027). A 18-gauge butterfly needle was then inserted in the ampulla of Vater, tied with Silkan thread. Through the needle, 10 ml of ice-cold collagenase (catalog number 11 213 865 001; Roche Applied Science) solution (1 mg/ml in Hanks' balanced salt solution (HBSS)-Hepes/glucose buffer (140 mM NaCl, 0.4 mM Na2HPO4, 4 mM NaHCO3, 5 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4·7H2O, 1.3 mM CaCl·2H2O, 2mM glucose, 10 mM Hepes, pH 7.2) was injected. The pancreas was surgically removed and placed in 10 ml of ice-cold collagenase solution and then transferred to a 37 °C water bath for 38 min. During this period, the sample was vortexed four times. Forty ml of ice-cold HBSS-Hepes/glucose buffer containing 4 mg/ml bovine serum albumin (isolation buffer) was then added, and the sample was centrifuged at 65 x g for 10 s. The next steps were all performed on ice. Part of the supernatant was aspirated so as to leave about 20 ml in the tube. The digested pancreas was then homogenized by six passages through a blunt 5-gauge needle. Twenty ml of isolation buffer was added and centrifuged at 65 x g for 10 s. Part of the supernatant was discarded (about 20 ml), and the remaining solution passed through the 5-gauge needle twice, complemented with 20 ml of isolation buffer and centrifuged as described above. Twenty ml of the supernatant was again discarded, and the remaining solution was filtered through two layers of sterile gaze. The filter was washed twice with 10 ml of isolation buffer. The collected solution was centrifuged at 500 x g for 10 s. The supernatant was discarded so as to leave about 5 ml in the tube. The sample was more tightly packed by a 10-s centrifugation at 400 x g. The supernatant was decanted, the pellet was gently resuspended in 20 ml of Histopaque 1077 solution (catalog number 10771; Sigma), layered carefully on 20 ml of isolation buffer, and spun in a Sigma 4K15 centrifuge at 1100 x g for 15 min (with setting 0 for the acceleration and the deceleration). The lower islet-containing phase (but not the pellet) was then harvested with a Pasteur pipette, and isolation buffer was added up to 40 ml. The sample was spun in a Sigma 4K15 centrifuge at 450 x g for 1 min (with setting 3 for the acceleration and the deceleration). The supernatant was discarded so as to leave about 5 ml in the tube, and isolation buffer was added up to 40 ml. The sample was then spun in a Sigma 4K15 centrifuge at 65 x g for 30 s (with setting 0 for the acceleration and the deceleration). The islets were washed once with 40 ml RPMI (catalog number 31870-025; Invitrogen) supplemented with 10% fetal calf serum, 10 mM Hepes, pH 7.4 (catalog number H3537; Sigma), 1 mM sodium pyruvate (catalog number S8636; Sigma), 2 mM glutamine (catalog number G7513; Sigma), 50 µM {beta}-mercaptoethanol (catalog number 31350-010; Invitrogen), and 1% penicillin/streptomycin solution (catalog number P0781; Sigma) and centrifuged as above. The supernatant was discarded so as to leave about 5 ml in the tube. The islets were then transferred to a 3-cm Petri dish, hand-picked (~500 islets/rat), and placed in a new 3-cm Petri dish. This procedure was repeated (5–10 times) until the majority of the exocrine cells was eliminated.

Dissociation of the islets was performed as follows. The islets were washed with 1 ml of HBSS-Hepes containing 1 mM EGTA and 5 mM glucose, resuspended in 300 µl of the same buffer, and incubated at 37 °C for 3 min. The islets were then pipetted up and down until loosely dissociated. The reaction was stopped by the addition of 1 ml of supplemented RPMI. The islets were washed again with 1 ml of supplemented RPMI, resuspended in the same medium at a concentration of about 20 islets/ml before being placed in 6-well plates (2 ml/well).

Mice islets were prepared in a similar manner as described for the rat islets with the following differences. Two ml of collagenase solution was injected through a 30-gauge needle, and the pancreas was transferred to 2 ml of the collagenase solution. The reaction was stopped by the addition of 30 ml of ice-cold HBSS-Hepes, centrifuged for 2 min at 400 x g at 4 °C, and washed three times with 40 ml of HBSS-Hepes, and the pellet was resuspended in 10 ml of HBSS-Hepes. Following filtration through the gaze, the islets were not layered on Histopaque but were directly resuspended in 10 ml of supplemented RPMI and hand-picked and dissociated as described above.



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  		FIGURE 3.
Insulinoma cells expressing fragment N are more resistant to cisplatin-induced apoptosis. A, {beta}TC-tet cells, {beta}TC-tet infected with empty viruses bearing or not bearing the neomycin (neo) resistance gene, and {beta}TC-tet infected with viruses expressing fragment N virus (with or without the neomycin resistance gene) were incubated with increasing concentrations of cisplatin for 24 h. The extent of apoptosis was then scored. This experiment has been repeated three more times with similar results. B, {beta}TC-tet cells and{beta}TC-tet cells stably expressing the Bcl-2 cDNA ({beta}TC-tet/Bcl-2 cells) were either not infected, infected with an empty virus, or infected with a lentivirus encoding fragment N. The viruses used here did not encode the neomycin resistance gene. The cells were then incubated with increasing concentrations of cisplatin for 24 h, and the extent of apoptosis was scored. This experiment has been repeated two more times with similar results.

 



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  		FIGURE 4.
Expression of fragment N in insulinoma cells leads to increased expansion and survival. {beta}TC-tet and {beta}TC-tet/Bcl-2 cells infected with the indicated lentiviruses were seeded in 6-well (3.7-cm) plates at a density of 50,000 cells/well. The cells in the wells were counted after the indicated periods of time in culture. The cells were left in the initial 2-ml culture medium until the end of the experiment.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Fragment N-expressing Lentiviruses—Fragment N can be cleaved, and its antiapoptotic properties abrogated, when caspase activity increases in cells (12). Fragment N cleavage can be prevented by introducing a point mutation in the caspase-3 recognition site at position 157 (13, 14). In this study, the terminology "fragment N" refers to the uncleavable form. Fragment N was introduced in {beta}TC-tet cells, expressing or not expressing Bcl-2 as a result of lentiviral infection (16), using two different types of fragment N-encoding lentiviruses. The first type expresses fragment N but no selection marker, whereas the second type bears a polycistron composed of fragment N followed by the neomycin resistance gene. Fig. 1 shows that both types of viruses induced the production of fragment N in cells. The levels of fragment N expression were, however, lower in cells infected with the neomycin resistance-encoding viruses compared with the cells infected with the other virus (Fig. 1). The two types of viruses provide, therefore, the opportunity to study the effect of high and low expression levels of fragment N on {beta} cell survival.



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  		FIGURE 5.
Expression of fragment N in insulinoma cells increases their resistance toward inflammatory cytokines. A, {beta}TC-tet and {beta}TC-tet/Bcl-2 cells stably infected with the indicated viruses were left untreated or incubated with 1000 units/ml of TNF{alpha}, interleukin-1{beta}, and interferon-{gamma}. Apoptosis was scored 1 day later. Results correspond to the mean ± S.D. of four independent experiments. B, {beta}TC-tet cells stably infected with empty viruses (control cells) or lentiviruses encoding fragment N were infected with empty lentiviruses or lentiviruses encoding a dominant negative mutant of Akt (DN-Akt). Forty-eight h later, the cells were incubated with 1000 units/ml TNF{alpha}, interleukin-1{beta}, and interferon-{gamma} for 24 h, and the extent of apoptosis was scored. Results correspond to the mean ± S.D. of two independent experiments performed in duplicate. The significance of the differences between the two cell types infected with the dominant negative Akt mutant and stimulated with cytokines was assessed by Student's t test. NS, not significant; *, p < 0.05.

 



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  		FIGURE 6.
Expression of fragment N in insulinoma cells confers long-term resistance toward inflammatory cytokines. Two hundred {beta}TC-tet and {beta}TC-tet/Bcl-2 cells stably infected with the indicated viruses were placed in p100 Petri dishes and left untreated or incubated with 1000 units/ml TNF{alpha}, interleukin-1{beta}, and interferon-{gamma} for 14 days. The colonies were then stained with Giemsa and counted. The upper panel depicts a representative experiment. The results presented in the lower panel are expressed as the percentage of clones obtained in the absence of cytokines (average ± S.D. of three independent experiments).

 
Fragment N has been shown to induce Akt activity and repression of the NF-{kappa}B pathway in HeLa cells (14). Fig. 2 shows that expression of fragment N in {beta}TC-tet cells, whether or not they express Bcl-2, also led to Akt activation coupled with a reduction of basal NF-{kappa}B activity. There is therefore no difference in the way fragment N regulates Akt and NF-{kappa}B in the different cell types tested so far.

Fragment N Increases the Resistance of {beta} Cells toward Various Stresses—We first determined the sensitivity of {beta}TC-tet cells to cisplatin, a genotoxic compound inducing apoptosis in many cell types, in the presence or absence of fragment N. {beta}TC-tet cells infected with empty viruses were as sensitive as control cells toward cisplatin-induced apoptosis (Fig. 3A). In contrast, a 4 times higher cisplatin concentration was required to kill the cells infected with fragment N-encoding viruses compared with control cells or cells infected with empty viruses (Fig. 3A). Cells infected with the fragment N-encoding virus bearing no selection marker were slightly more resistant toward cisplatin compared with cells infected with fragment N-encoding virus bearing the neomycin-resistant gene (Fig. 3A). This is probably due to the lower expression of fragment N in the former cells compared with the latter (Fig. 1).



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  		FIGURE 7.
Fragment N increases the resistance of growth-arrested {beta}TC-tet cells toward inflammatory cytokines without affecting their glucose-induced insulin secretion. {beta}TC-tet and {beta}TC-tet/Bcl-2 cells stably infected with the indicated viruses were incubated with tetracycline (1 µg/ml) for 5 days to repress the expression of the large T antigen and to promote growth arrest (16). Expression of the large T antigen was assessed by Western blot (A). The cells were then treated as described in the legend to Fig. 5 to assess their sensitivity toward cytokine-induced apoptosis (B). The insulin content of the growth-arrested cells was also determined (C). Results are expressed as the percentage of the insulin content in control cells and represent the mean ± S.D. of triplicate determinations. Alternatively, the growth-arrested cells were incubated in 2 mM glucose-containing medium for 1 h and switched to a 2 or 20 mM glucose-containing medium for 35 min. The amount of insulin secreted was then measured by an enzyme-linked immunosorbent assay (D). Results are expressed as a percentage of cellular insulin content and correspond to the mean ± S.D. of triplicate determinations.

 
We next assessed whether the protective effect of fragment N could synergize with those of Bcl-2, another antiapoptotic molecule that has been shown to increase the resistance of {beta}TC-tet toward hypoxia- and cytokine-induced apoptosis (16). As expected, the presence of Bcl-2 rendered {beta}TC-tet cells about 2.5 times less sensitive to cisplatin-mediated cell death (compare the EC50 of the red lines in Fig. 3B). The Bcl-2-expressing cells could be rendered 4 times more resistant toward cisplatin in the presence of fragment N (Fig. 3B, right). Therefore, the combined presence of Bcl-2 and fragment N rendered {beta}TC-tet cells 10 times more resistant to cisplatin.

We next determined the capacity of {beta}TC-tet cells to cope with cell crowding as a means to assess their resistance toward a degradation of their trophic and growth conditions. Control {beta}TC-tet cells grew more or less exponentially up to 7–8 days, and their population collapsed thereafter within the following 3 days (Fig. 4). The presence of Bcl-2 slightly delayed the collapse of the culture by 1–2 days. In contrast, cells expressing high or low levels of fragment N (i.e. cells derived after infections with non-neomycin-encoding versus neomycin-encoding viruses (see Fig. 1)) appeared to grow faster and collapsed 2–3 days later and at cell densities that were 2–4 times higher compared with control cells. These results indicate that the presence of fragment N allows cells to grow and survive longer in degraded environments.

To assess the resistance of {beta}TC-tet cells to an inflammatory environment, {beta}TC-tet cells were incubated with a mixture of inflammatory cytokines. Noninfected cells or cells infected with empty viruses encoding or not encoding the neomycin resistance gene displayed a ~3-fold increase in the apoptotic rate in response to the cytokine mixture (Fig. 5A). In contrast, when the cells were infected with either type of viruses encoding fragment N, the cytokines only increased apoptosis by 1.5–1.6-fold. The presence of Bcl-2 did not protect cells from apoptosis in response to the concentrations of cytokines used here. Akt activity was required for the protection conferred by fragment N, because a dominant negative mutant of Akt prevented fragment N from inhibiting cytokine-induced apoptosis (Fig. 5B).

It could be argued that fragment N may only confer a temporary protection (such as the one seen in Fig. 5A) but no long term survival advantage. To assess this point, {beta}TC-tet and {beta}TC-tet/Bcl-2 cells expressing or not expressing fragment N were placed in Petri dishes and allowed to grow for about 2 weeks in the absence or in the continuous presence of inflammatory cytokines. As shown in Fig. 6, the presence of inflammatory cytokines diminished the ability of {beta}TC-tet to form colonies by a factor of about 10 regardless of whether or not the cells expressed Bcl-2. In contrast, inflammatory cytokines diminished the capacity to form colonies by less than 2-fold in {beta}TC-tet cells expressing fragment N. The co-expression of fragment N with Bcl-2 did not appear to further increase the capacity of {beta}TC-tet cells to generate colonies (Fig. 6). Altogether, these results demonstrate that fragment N confers long term protection against the proapoptotic actions of inflammatory cytokines when other antiapoptotic proteins (e.g. Bcl-2) cannot.

The {beta}TC-tet cells express the large T antigen, allowing them to proliferate. Although it has been recently demonstrated that differentiated {beta} cells have the capacity to proliferate to compensate for a loss in {beta} cell mass (25), mature {beta} cells probably have a low proliferation activity. {beta}TC-tet cells can be growth-arrested by repressing the expression of the large T antigen with tetracycline (Fig. 7A) and in this state can secrete insulin with a normal glucose dose dependence (20). We therefore determined whether fragment N would also protect {beta}TC-tet when growth-arrested. As shown in Fig. 7B, the presence of fragment N protected growth-arrested {beta}TC-tet cells as efficiently as it did in proliferating {beta}TC-tet cells. Importantly, the presence of fragment N altered neither the insulin content of the cells (Fig. 7C) nor their ability to secrete insulin in response to increased glucose concentrations (Fig. 7D). Altogether, these results indicate that fragment N potently protects {beta} cells from a variety of stress and noxious stimuli without interfering with their insulin secretion capacity.

Fragment N Protects Primary Islet Cells from Cytokine-induced Death—To determine whether fragment N also mediates protection in primary islet cells, mouse and rat islets of Langerhans were isolated, dissociated, infected with empty lentiviruses or virus encoding fragment N, and then incubated or not with inflammatory cytokines. Fig. 8 shows that fragment N potently protected islet cells from both species from cytokine-induced apoptosis (Fig. 8A) and that this correlated with increased levels of phosphorylation of Akt at its activation sites (Fig. 8B). These results indicate that fragment N activates Akt and promotes survival in both transformed and primary {beta} cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Elimination of pancreatic {beta} cells by apoptosis is a culminating event leading to diabetes. The development of tools favoring {beta} cell survival in patients is therefore of critical importance to delay or prevent the development of the disease (26). Moreover, compounds that increase {beta} cell survival would be extremely useful in islet transplantation procedures, such as the Edmonton protocol, to increase the yield of islet cell production from diseased donors and to ameliorate the rate of successful engraftment of the pancreatic islets in the host (26).

In the present study, we describe a new strategy to protect insulin-secreting cells based on the expression of an antiapoptotic N-terminal Ras-GAP fragment called fragment N. Efficient protection of the insulin-secreting {beta}TC-tet cell line against genotoxins, degraded cellular environments, and inflammatory cytokines was achieved even with the lowest cellular expression of fragment N tested. It seems therefore unnecessary to strongly express fragment N in order to protect insulin-secreting cells.

The potencies of fragment N and Bcl-2 to protect insulinoma cells were compared. Expression of both proteins in cells was achieved through infection with lentiviral vectors (this study) (16). The same methodology was therefore used to express these two proteins in the insulin-secreting cell line. In these conditions, it was observed that fragment N protected cells more efficiently against a variety of noxious stimuli than Bcl-2. The two proteins, however, clearly induced an additive protection signal when combined (e.g. against cisplatin-induced apoptosis or to counteract the negative effects of degraded environments). Strategies using two antiapoptotic molecules are therefore not necessarily mutually exclusive and might in fact confer additional levels of {beta} cell protection.



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  		FIGURE 8.
Fragment N protects primary islet cells from cytokine-induced apoptosis. Freshly dissociated rat and mouse islets were infected with empty lentiviruses or lentiviruses encoding fragment N. Twenty-four hours later, the cells were incubated or not with inflammatory cytokines (1,000 units/ml TNF{alpha}, 1,000 units/ml interleukin-1{beta} and 50 units/ml interferon-{gamma}) for an additional 24-h period before scoring apoptosis (A). Results correspond to the mean ± S.D. of 3–4 independent determinations. The significance of the differences between cell types incubated or not with cytokines was assessed by Student's t test (NS, not significant; **, p < 0.01). Alternatively, rat islet cells were lysed, and the levels of active Akt and total Akt were assessed by Western blot analysis as described in the legend to Fig. 2A (B).

 
A very potent antiapoptotic mediator is the Akt kinase (27). Akt is able to stimulate the NF-{kappa}B pathway (8, 14, 28), and in some cell types, Akt-induced NF-{kappa}B activation is required for cell survival (8). In {beta} cells, however, NF-{kappa}B stimulation can be detrimental (3, 10, 11, 29, 30). Therefore, in contrast with many cell types, expression of active Akt in {beta} cells and a concomitant NF-{kappa}B activation could favor apoptosis. This has indeed been confirmed in vivo in mice expressing an Akt1 transgene under the control of the insulin promoter (31).

In HeLa cells, the N-terminal fragment of RasGAP generated following its partial cleavage by caspase-3 induces Akt but prevents Akt from activating the NF-{kappa}B pathway (14). We show here that fragment N does the same in insulin-secreting cells. Expression of fragment N in pancreatic {beta} cells would therefore allow Akt to fulfill its antiapoptotic and proliferative functions and, at the same time, repress the potentially detrimental NF-{kappa}B-inducing activity of Akt (10, 11). Our results indeed demonstrate that expression of fragment N in the {beta}TC-tet insulinoma cell line confers long term protection from various adverse conditions and apoptotic stimuli, including inflammatory cytokines that are believed to be the pathophysiological mediators of {beta} cell death leading to diabetes (3). Fragment N did not, however, affect the insulin secretion capacity of {beta}TC-tet cells. This fragment represents therefore a potential therapeutic tool to protect {beta}-cells in diabetogenic conditions without compromising their physiological properties.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grant 3100-066797/1 and the Botnar Foundation (Lausanne, Switzerland). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Cellular Biology and Morphology, Biology and Medicine Faculty, University of Lausanne, Bugnon 9, 1005 Lausanne, Switzerland. Tel.: 41-21-692-5123; Fax: 41-21-692-5255; E-mail: Christian.Widmann{at}unil.ch.

2 The abbreviations used are: TNF{alpha}, tumor necrosis factor {alpha}; PBS, phosphate-buffered saline; HA, hemagglutinin; HBSS, Hanks' balanced salt solution. Back


   ACKNOWLEDGMENTS
 
We thank Guy Niederhauser and Gilles Dubuis for technical assistance and Dr. Peter Clark for suggestions and comments. We also thank Dr. Isabelle Decosterd and Marie Pertin for help with rat manipulations.



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 DISCUSSION
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